Expansions of simple DNA repeats are implicated in more than thirty hereditary neurological and developmental disorders in humans. This proposal is devoted to the molecular mechanisms responsible for repeat expansions. My lab was the first to show that expandable repeats stall replication fork progression in vivo in a length-dependent manner, reminiscent of their instability pattern in humans. This phenomenon was observed in all experimental system studied from bacteria to mammalian cells and subsequently confirmed by many other labs. This led us and others to formulate the replication model for repeat expansions stipulating that repeats could be added as the replication fork attempts to escape from the repetitive trap. During the previously funded period, we have developed first-of-a-kind system to detect large-scale repeat expansions in yeast, S. cerevisiae. Importantly, many features of repeat expansions seen in this system closely matched those observed in human pedigrees. Furthermore, genetic screening at the whole-genome level revealed that repeat expansions occur primarily during their replication in the process likely involving DNA template switching. We also found that while transcription is not necessary for the repeat expansions, it seems to strongly contribute to the process. We plan to continue these studies of the molecular mechanisms of repeat expansions in yeast and mammalian cells. We will study the role of transcription and transcription-replication interplay in the expansion process by developing experimental systems, in which transcription of the repeat is known and can be controlled. These new experimental systems enable us to carry out side-by-side comparisons of the expansion mechanisms for various DNA repeats. Our current working hypothesis is that expansion pathway for each individual DNA repeat can depend on a delicate balance between its propensity to form DNA hairpins versus its propensity to deviate the replication fork progression. This idea will be ascertained by comparing expansion rates and scales for different repeats in the same experimental settings, as well as by carrying out comparative genetic analysis of different repeat expansions via candidate gene approach. We will also search for genetic modifiers of repeat expansions by conducting an unbiased mutagenic screen followed by whole-genome sequencing to identify causative mutations. To study large-scale repeat contractions in yeast, we will develop a system utilizing a selectable cassette that contains disease-sized repeats in the intron of the SUP4-o suppressor tRNA allele. We will then identify genes affecting the contraction process by carrying out a screen with the yeast mini- transposon genomic library. Finally, we will continue working on a selectable system for the analysis of repeat instability in cultured mammalian cells. It is based on the integration of a cassette, which carries the HyTK gene under the control of the FMR1 promoter with carrier-length (CGG)n repeats in its 5'UTR, into a unique genomic site of the murine erythroid leukemia or human HEK-293 cells. We will used this approach to detect repeat expansions and repeat-mediated mutagenesis, and to unravel the mechanisms responsible of the instability using siRNA or shRNA against the candidate genes identified in our yeast screens. The long-term goal of this proposal is to understand the molecular mechanisms responsible for repeat instability in humans. In the long run, we hope to understand these mechanisms in details sufficient to propose new therapeutic strategies for treatments of these debilitating human diseases.